Animal Models in Alcohol Research

Animal models are important tools in the study of alcohol use, abuse, and dependence because they allow researchers to use methods that cannot be used with human subjects. Animal models have been developed to study various aspects of alcohol use and dependence, including alcohol-seeking behavior, alcohol-related organ damage, tolerance to alcohol, and physical dependence on alcohol. Because animal models can be genetically manipulated, they are also valuable for research into the genetic determinants of alcoholism. Issues surrounding the use of animal models in alcohol research include the species of animal used, the method of alcohol administration, and the model’s face and predictive validity.

B ecause of ethical concerns and experimental difficulties in studyi n g alcoholism in humans, a substantial portion of re s e a rch on the topic of alcohol intoxication and dependence has used nonhuman animals as experimental models. A model, in this sense, refers to something that is used to help v i s u a l i ze that which cannot be dire c t l y o b s e rved. In other words, by using experi m e n t a l animals, scientists are attempting to dissect the complex disorder of alcoholism, in part by breaking it down into its component behaviors and studying the determinants of those behaviors.
The behaviors that characterize alcoholism in humans, according to the criteria for diagnosing alcoholism outlined in the Diagnostic and Statistical Manual of Mental Disord e r s , Fo u rth Edition (DSM-IV) ( A m e r i c a n Ps yc h i a t r i c Association 1994), include the following: (1) tolerance, or the need for increased amounts of alcohol to obtain the desire d effects; (2) withdrawal symptoms after discontinuation of alcohol use; (3) taking alcohol in large amounts over periods longer than initially intended; (4) persistent desire or unsuccessful efforts to decrease alcohol use; (5) spending a great deal of time acquiring alcohol; (6) re d u c i n g i m p o rtant social and occupational activities because of alcohol use; and (7) continued use despite a re c u r re n t physical or psychological problem associated with alcohol use.
One system (A) is a model for another system (B) if the study of A furthers the understanding of B, re g a rdless of any causal connection between them ( K a p l a n 1964; McClearn 1988). For the model to be efficient, system A should be simpler than system B (McClearn 1988).
T h e re f o re, the animal models that are most commonly used in alcohol re s e a rc h h a ve been designed in an attempt to understand, at the physiological, biochemical, or molecular level, the basis for a particular behavior that is believe d to be an analog of a behavior associated with human alcoholism.
This article discusses the adva n t a g e s of using animal models, especially in alcohol re s e a rch, presents issues re l a t e d to the development and use of animal models of alcoholism, and describes various animal models that have been d e veloped to study aspects of alcoholism. We have focused, for the most part, on animal models of exc e s s i ve alcohol intake, because this is the key factor that leads Animal Models in Alcohol Re s e a rc h Boris Tabakoff, Ph.D., and Paula L. Hoffman, Ph.D.

Animal models are important tools in the study of alcohol use, abuse, and dependence because they allow researchers to use methods that cannot be used with human subjects. Animal models have been developed to study various aspects of alcohol use and dependence, including alcohol-seeking behavior, alcohol-related organ damage, tolerance to alcohol, and physical dependence on alcohol. Because animal models can be genetically manipulated, they are also valuable for research into the genetic determinants of alcoholism. Issues surrounding the use of animal models in alcohol research include the species of animal used, the method of alcohol administration, and the model's face and predictive validity. KE Y W O R D S: animal
model; scientific model; research; AOD (alcohol or other drug) dependence; AOD tolerance; body part; animal selectively bred for AOD preference; quantitative trait locus; route of administration; theory of AODU (AOD use, abuse, and dependence) to organ damage and alcohol dependence. The crucial question to be a n s we red using these models is the foll owing: Why do some people consume alcohol in quantities that are injurious to themselves and to those around them? We also describe in a more limited way c e rtain models that are used to determine how alcohol produces damage to various organs. These descriptions are included as examples of models that can be used to understand the re s u l t s o f e xc e s s i ve alcohol drinking, rather than the mechanisms which motivate people to drink alcohol.

Advantages of Animal Models
Animal models allow re s e a rchers to use methods that would be unethical with human subjects. In some cases, using humans in alcohol re s e a rch (and in re s e a rch on other drugs of abuse) raises specific ethical issues, such as the risk i n vo l ved in administering an addictive d rug to humans and the related risks of accidents and of medical and psychological consequences. Clearly, these risks can be circ u m vented in animal studies. Because of the risks inherent in human alcohol studies, as well as the limitations imposed when human subjects are used, animal model studies h a ve been, and continue to be, inva l uable for addressing the basic questions of alcohol re s e a rc h .
T h e re are also scientific reasons to use animal models. Although cells and tissues can be used for biochemical and molecular biological studies, there is no way to relate the results of these studies d i rectly to any particular behavior.
T h e re f o re, re s e a rch into determinants of behavior can best be carried out in experimental animals. A hallmark and an advantage of animal re s e a rch, especially re s e a rch into complex disord e r s such as alcoholism, is that it has the effect of simplifying complex behaviors by producing models that are re l e va n t to the human situation.
Both human and animal studies indicate that genetic factors play a ro l e in the development of alcoholism, leadi n g re s e a rchers to focus on identifying genes associated with alcoholism or a p redisposition to alcoholism. Re s e a rc h in this area has benefited from the use of genetic engineering techniques.

Validity of Animal Models
By using animal models to study human d i s o rders, we are implicitly acknow l e d ging the evo l u t i o n a ry relationship betwe e n humans and other animals. There is abundant evidence that various ve rt ebrate (and even inve rtebrate) species h a ve similar biochemical and physiological systems, although sometimes these systems have different purposes. In addition, embryonic development in animals is similar to that of humans, a l l owing the study of the effects of alcohol and other drugs on this deve l o p m e n t . Ne ve rtheless, animal species differ, and the re l e vance of results obtained using animal models to the human situation may depend on the species chosen. In addition, animal models may have either face validity (i.e., they mimic some aspect of the human condition) or pred i c t i ve validity (i.e., results obtained with the animal model are pre d i c t i ve of alcohol's actions or of treatment efficacy in humans). We would expect that animals most closely related to humans genetically or evolutionarily (e.g., nonhuman primates), would provide the best face va l i d i t y. Howe ve r, studies of alcohol effects in nonhuman primates a re ve ry expensive and technically difficult. Fo rt u n a t e l y, many rodent models h a ve been developed to study both the causes of human diseases and the efficacy of treatments (e.g., mouse models for anticonvulsant drugs, rat models for a n t i d e p ressant drugs). In these cases, the models have pre d i c t i ve validity (i.e., medications that are effective in the model are also effective in humans).

Animal Models of Alcoholism: Dependence, Organ Damage, and Behavioral Consequences
Animal models of many aspects of human alcoholism have been deve l o p e d . In these models, which primarily use rodents such as mice and rats, the re s e a rcher usually controls alcohol intake. Alcohol may be fed to the animals in a liquid diet as their sole source of nutrition, administered by a tube implanted into the stomach (i.e., intragastric administration) or by injection, or administere d t h rough inhalation in specially designed chambers. The goal of these types of models is to generate the adaptive changes in the brain that are associated with the d e velopment of tolerance to and physical dependence on alcohol, both of which can be assessed in the animals.

Dependence and Tolerance
The characteristics of alcohol dependence and tolerance in mice and rats a re similar to those exhibited by humans. By measuring tolerance-or d e p e n d e n c e -related changes in brain b i o c h e m i s t ry and gene expression in animal models, re s e a rchers have made p ro g ress in identifying the neuro c h e m ical systems associated with alcohol tolerance and physical dependence, and h a ve introduced a novel concept indicating that alcohol withdrawal can produce brain damage. Howe ve r, most of these models lack a key element of the human situation because the re s e a rc h e r, rather than the animal, determines the alcohol intake. In addition, enviro nmental elements play a role in tolerance. A person may exhibit tolerance to the effects of alcohol while drinking in a favorite restaurant or bar, but at the same blood alcohol concentration, may not exhibit tolerance when in a completely novel environment, such as when driving on an unfamiliar highw a y. Although a number of studies h a ve tried to model enviro n m e n t a l

Re s e a rch into d e t e rminants of behavior can best be carried out in experimental animals.
influences on the expression of alcohol tolerance or physical dependence in animals, the measure of enviro n m e n t a l influence is not a major component of many studies of tolerance, dependence, or other consequences of alcohol ingestion.

Organ Damage
Animal models have also been used to examine the mechanisms by which alcohol produces organ damage (Lieber et al. 1989). Both rat and baboon models have proven useful for studying the toxic effects of alcohol on the live r and how these effects va ry with nutrition. Lieber and colleagues (1965) fed alcohol to rats as part of a nutritionally adequate liquid diet containing re l at i vely large amounts of fat, but no live r lesions more adva n c e d t h a n fatty live r could be produced by this method. Tsukamoto and colleagues (1986) and French and colleagues (1986) p ro d u c e d m o re seve re liver damage in rats with continuous intragastric administration o f alcohol and a nutritionally defined l ow-fat liquid diet, and this damage was increased by increasing the fat content of the diet. This method pro d u c e d a model of alcoholic liver disease in rats that is more comparable to the disease that occurs in humans. Lieber a n d DeCarli (1974) also used a liquid diet feeding technique with baboons. In this model, the biochemical and morphologi c a l changes in the live r, including cirrhosis, mimic those seen in human a l c oholics, although alcoholic hepatitis is not observed. Di s c re p a n c i e s in the ability to produce alcoholic liver disease using this model (e.g., Rogers et al. 1981;Mezey et al. 1980Mezey et al. , 1983 may be due to the nutrient value of the diets used or to the fact that some studies used a small number of animals (Lieber and DeCarli 1991). These findings illustrate the importance of the choice of species for animal studies using alcohol. The baboon model, which uses nonhuman primates that are genetically or e volutionarily close to humans and h a ve a long life span, may be more i n f o r m a t i ve when considering the full s p e c t rum of liver damage in humans.
The mechanism by which alcohol induces disorders of the heart muscle ( c a rd i o m yopathies) has also been studied in animal models. This re s e a rch has sh own that alcohol acutely decreases the synthesis of contractile proteins in the h e a rt and has suggested mechanisms for this decrease. Such studies have also explained the mechanisms by which c h ronic alcohol exposure damages heart muscle (Patel et al. 1997). Re c e n t l y, a chicken model of alcohol-induced card i o m yopathy has been developed, which appears to mimic the human condition m o re closely, providing a new tool to study the mechanism of alcohol's effects ( Morris et al. 1999). These models add significantly to human epidemiological studies, because re s e a rchers can assess the mechanisms of alcohol-induced heart damage while controlling confounding factors and the amount of alcohol admini s t e re d or ingested.
Human studies have found low to moderate alcohol intake to have a benefic i al effect on the heart. Animal models h a ve been developed recently to study the mechanism of this effect (e.g., Miyamae et al. 1998).

Alcohol-Related Behavior
Re s e a rc h e r s h a ve also attempted to cre a t e animal models of various human alcoholrelated behaviors. For example, many studies have investigated alcohol-associated a g g ression in rats and mice (e.g., Fish et al. 1999;Mi c zek et al. 1997), prov i d i n g evidence of the physiological and neurochemical changes associated with incre a s e d a g g ression. These findings led to the deve lopment of therapies, such as selective s e rotonin reuptake inhibitors (compounds that prolong the activity of the neurotransmitter serotonin), which may be e f f e c t i ve in reducing the incidence of alcohol-associated violence. Howe ve r, the d rugs used for management of violence h a ve only a nominal effect in re d u c i n g human alcohol intake (Hoffman and Tabakoff 1999).

Animal Models of Alcohol Intake
The studies described above re p re s e n t attempts to generate animal models displaying face validity for alcohol-induced pathological changes in humans. Animal models of alcohol intake have also been d e veloped, including a number of animal models of exc e s s i ve alcohol intake (Weiss and Koob 1991). These models of alcohol-seeking behavior attempt to demonstrate the re i n f o rcing (pleasurable) pro p e rties of alcohol, which are thought to play a key role in human alcohol use. Many of these models are embraced because they appear to have face va l i d i t y, but this may be misleading; after all, it is difficult to identify the impetus for a behavior in a rodent or a nonhuman primate and to fully re p resent the human condition. These models m a y, howe ve r, have pre d i c t i ve va l i d i t y, and may also be valuable for determining the neurochemical and molecular pathways that contribute to alcohol use.
One method of assessing alcohol intake uses animals that are given a choice between alcohol and another fluid. For example, in the two-bottle choice method, rats are allowed an unrestricted choice between alcohol and water 24 hours per day. Alternative l y, access to alcohol, either alone or with a choice of another fluid, such as water, can be restricted to a certain period of time during the day. Se veral appro a c h e s h a ve been used to produce a re l i a b l e demonstration of vo l u n t a ry alcohol drinking. These include alcohol acclimatization (i.e., providing gradually i n c reasing alcohol concentrations), taste adulteration (i.e., addition of swe e t e n e r s to the alcohol solution), and the use of prandial models, which take adva n t a g e of the postmeal drinking seen in rats.
In all of these models, animals are a l l owed to consume alcohol vo l u n t a r i l y. If nothing else, these models have show n that heterogeneous populations of animals, like humans, display a large range of alcohol consumption and that alcohol is consumed more avidly if it is contained in sweetened solutions (i.e., these models h a ve face validity). These studies have also shown that genetic manipulation by i n b reeding or selective breeding can produce animals displaying ve ry defined (either high or low) alcohol pre f e re n c e .
An inherent limitation of the twobottle choice method and others of this type is that it is difficult to use them to demonstrate the animal's motivation to obtain alcohol. Mo t i vation is demonstrated, howe ve r, in operant models (i.e., models in which the animal must p e rform a certain task to re c e i ve alcohol) of alcohol intake. In these models, an animal is trained to press a lever and re c e i ve alcohol, generally by the oral route. By adjusting the number of leve r p resses needed to re c e i ve the alcohol "rew a rd," one can assess how hard an animal will "w o rk" to re c e i ve the alcohol. In some instances, the alcohol is a d m i n i s t e red via injection directly into the stomach through surgically implanted tubes (i.e., intragastric selfadministration). This method has been used to avoid the influence of taste (i.e., to assure that alcohol is being administered by the animal for its pharmacological pro p e rties). The alcohol may also be self-administere d d i rectly to the brain. Using this pro c ed u re, re s e a rchers can identify the brain regions invo l ved in alcohol's re i n f o rc i n g effects and minimize confounding factors such as metabolism (Meisch and L e m a i re 1993).
One of the most successful operant models for inducing re l a t i vely high leve l s of oral alcohol intake by rodents uses a s weet solution (e.g., saccharin) to introduce animals to alcohol, after which the concentration of sweetener is gradually reduced. This pro c e d u re produces re l i a b l e operant responding for alcohol within a reasonable length of time (i.e., weeks) and can generate blood alcohol levels high enough to affect the animal's behavior. A modification of this model has been described in which food-and water-sated rats that had been operantly trained to administer alcohol orally we re allowed to obtain water or alcohol by responding on one of two levers. This paradigm a d d resses several key issues re g a rding alcohol re i n f o rcement: (1) alcohol intake is maintained by pharmacological motivation, rather than factors related to appetite or thirst, and (2) alcohol changes and maintains the leve r -p ressing behavior, which functions to provide alcohol to the animal. The maintenance of leve r -p re s sing behavior is interpreted as an indication that alcohol is functioning as a re i nf o rcer (Weiss and Koob 1991). Using this model, certain rats have been shown to display a significant pre f e rence for alcohol over water and to achieve high blood alcohol concentrations.
Operant alcohol self-administration has not only been used to assess the re i n f o rcing effect of alcohol, but also to model the craving for alcohol experienced by abstinent alcoholics. When animals h a ve been drinking alcohol re g u l a r l y and are then subjected to a period of f o rced abstinence from alcohol, they s h ow a reliable increase in alcohol intake w h e n alcohol is again made available (i.e., the alcohol deprivation effect). Whether this apparently enhanced motivation to ingest alcohol is an accurate model of craving in humans is not clear. Howe ve r, this model does have significant pre d i ct i ve validity; the drugs now used to reduce craving and relapse in humans (e.g., acamprosate and naltre xone) can also block the increased re s p o n d i n g associated with the alcohol depriva t i o n effect in animal models.
Another method used to assess the re i n f o rcing pro p e rties of alcohol is conditioned place pre f e rence. For this proc e d u re, the animal re c e i ves alcohol and is then placed in a distinctive enviro nment. The animal associates the effect of alcohol with that environment. If the effect of alcohol is pleasant (re i n f o rcing) to the animal, it will later choose the distinctive environment over another e n v i ronment when given a choice. In contrast, an animal that finds the effect of alcohol ave r s i ve will spend less time in the alcohol-associated enviro n m e n t . Mice have been found to demonstrate a conditioned place pre f e rence for alcohol in a number of studies, although an alcohol place pre f e rence is more difficult to demonstrate in rats and seems to re q u i re pre e x p o s u re of the rats to alcohol. Rats are more likely to show a version to alcohol in this model. The reason for this difference is not know n , but again illustrates the importance of the choice of animal models for mimicking aspects of human behavior.
Drug discrimination pro c e d u re s p rovide another method for ascert a i ning alcohol's pharmacological pro p e rties and sites of action. In the simplest application of the pro c e d u re, animals a re trained to respond for a food rew a rd using a particular lever when alcohol (i.e., the re f e rence drug) is a d m i n i s t e red and another lever when water is administered. The animal is then given an agent (i.e., a test dru g ) that is known to act at a specific re c e ptor (i.e., a binding site for a specific brain chemical) and is allowed to re s p o n d o n either leve r. If the animal perc e i ves that the effect of the test drug is similar to that of alcohol, the animal will respond with the alcohol-associated leve r. Although t h e receptor sites identified with this model may or may not play a role in mediating the re i n f o rcing effect of alcohol, this model can be used to identify targets for the development of therapies to interf e re with various actions of alcohol.
Most or all of the animal models outlined above probably do not have face va l i d i t y, but many have substantial p re d i c t i ve va l i d i t y. Animal models with a great degree of face validity are primarily nonhuman primates, living in a social situation, that have access to alcohol. These animals are expensive to maintain and re q u i re substantial expertise on the part of the inve s t i g a t o r, but the paradigm has the potential to provide re l e vant behavioral, genetic, pharmacological, and neurochemical information. For example, personality characteristics and the influence of re a r i n g experiences on alcohol consumption h a ve been studied in rhesus monkeys living in social groups, and these monkeys could also be evaluated for neurochemical characteristics, such as the activity of neurotransmitter systems in the brain ( Higley and Linnoila 1997). The results of studies using these models appear to provide important parallels to the human situation and should h a ve considerable pre d i c t i ve validity as well as face va l i d i t y.

Selective Breeding and Other Genetic Models
It is generally accepted that there are both environmental and genetic influences, and interactions between these factors, on the development of human alcohol dependence (i.e., alcoholism). The strong evidence for a role of genetics in human alcoholism (e.g., twin and adoption studies; alcoholism risk in individuals with family histories; subtypes of alcoholism) has led to a substantial e f f o rt to identify "a l c o h o l i s m -re l a t e d" genes by using genetic animal models.
For this type of re s e a rch, it is desirable for investigators to use animals whose genetic make-up (i.e., genome) has been well characterized, as well as animals for which a known re l a t i o n s h i p exists between the organization of the animal genome and the human genome. The mouse best meets these criteria, c u r re n t l y, but the genomic information on other animal species (e.g., rat or monkey) is being rapidly accumulated. When the genomic information on the other species is available, it will contribute to the enhanced growth of genetically focused animal models. Mouse models of various alcohol-related behaviors h a ve been successfully used to identify p o rtions of the mouse genome associated with these behaviors and, thus, to indicate analogous regions of the human genome that may be associated with the same behaviors (e.g., alcohol's effects on coordination). Although mice are generally used as models for alcohol re s e a rch, a recent study found that the organization of the human genome is closer to that of the chicken than to that of the mouse (Bu rt et al. 1999). This finding raises the question of whether scientists should be modeling alcoholrelated behaviors in chickens rather than mice. Di f f e rences in behavior betwe e n avians and mammals, howe ve r, may o u t weigh this possible advantage. Fo r example, chickens would not be good models for studying infants' genetic sensitivity to brain damage from alcohol ingested through mother's milk.
The first question that arises when s e a rching for genetic determinants for a l c o h o l i s m -related behaviors is, which a l c o h o l -related behaviors are the most re l e vant? The behaviors that have been most widely studied include sensitivity to alcohol's effects; changes that occur in response to chronic alcohol exposure , such as tolerance, physical dependence, and sensitization; and alcohol "p re f e rence," or intake.
Se l e c t i ve breeding produced some of the earliest genetic models of alcoholrelated behaviors and is still generating i m p o rtant information (Crabbe and Belknap 1992; Mc Bride and Li 1998). In this technique, mice or rats are bre d to create lines of animals that are sensit i ve or insensitive to a particular effect of alcohol. Most of the selectively bre d lines currently available originate fro m genetically heterogeneous foundation populations whose individuals we re s c reened for sensitivity to alcohol's effects. Breeding pairs are chosen for extre m e sensitivity or insensitivity, and pro d u c e offspring that are also screened and s e l e c t i vely bred. This process is continued for several generations, until highly s e n s i t i ve and insensitive lines are produced. Theore t i c a l l y, if inbreeding is a voided or minimized, the genes that influence the selected trait will be "f i xe d" while genes unrelated to the selected trait will be randomly distributed in the selected lines. There f o re, if the selected lines differ in a biochemical or behavioral trait other than the one for which they have been selected (i.e., a "c o r related trait"), it can be concluded that a common set of genes influences the two traits.
Se l e c t i ve breeding has been u s e d e x t e n s i vely to study alcohol-re l a t e d behaviors, and lines have been bre d that differ in sensitivity to the hypnotic effect of alcohol (e.g., long-sleep and s h o rt-sleep mice); the hypothermic effect of alcohol (e.g., COLD and HOT mice); the locomotor stimulatory effect of alcohol (e.g., FAST and SLOW mice); alcohol withdrawal seizures (e.g., withdrawal seizure -p rone [WSP] and re s i s t a n t [WSR] mice); and acute functional alcohol tolerance (a measure of tolerance to alcohol that occurs within one testing session as opposed to tolerance deve lopment that occurs over several days of alcohol treatment and testing) (e.g., HAFT and LAFT mice). In addition, at least five lines of rats that differ in their alcohol intake (pre f e rence) have been bre d . In all cases, animals have been bred bidire c t i o n a l l y (i.e., pairs of selected lines we re generated that displayed high and l ow responses to the particular effect of alcohol being studied, or high and low p re f e rence for alcohol), and in most cases replicate lines have been bred to a l l ow rapid verification of any differences between one pair of selected lines. Selected lines have provided a great deal of information re g a rding the genetic and biochemical basis of alcohol-re l a t e d responses. In part i c u l a r, rat lines selected for alcohol pre f e rence have been show n to ingest alcohol for its pharmacological p ro p e rties, to differ in correlated traits such as sensitivity and tolerance to va r ious alcohol effects, and to prov i d e good pre d i c t i ve validity for identifying therapies to reduce alcohol intake ( Mc Bride and Li 1998).
T h e re are a number of caveats associated with the selective bre e d i n g a p p roach. For example, some inbre e d i n g is unavoidable because of the re l a t i ve l y small population size in selective bre e ding studies, resulting in the fixation of genes unrelated to the selected trait. The estimation of genetic corre l a t i o n s , based on correlated responses to selection, is subject to a number of statistical as well as genetic considerations (Cr a b b e et al. 1990). It is also necessary to consider whether the behavior being selected (e.g., sensitivity to alcohol-induced loss of ability of an animal to right itself when placed on its back ["righting re f l e x" ] ) reflects primarily the effect of alcohol on the central nervous system, or whether the behavior may also be influenced by other factors, such as the mouse's ove rall coordination, body weight, or body fat. A coro l l a ry of this issue is whether the behavior (e.g., the anesthetic effect of alcohol) that is measured in the selected species is re l e vant to human m o t i vation for ingesting alcohol.
Other genetic models of alcoholrelated behaviors include inbred strains, recombinant inbred strains, and transgenic/knock-out mice (Go r a - Maslak et al. 1991;Wehner and Bowers 1995). An inbred strain is usually derived fro m s u c c e s s i ve brother-sister matings for at least 20 generations, resulting in a genetically identical group of animals.

Se l e c t i ve bre e d i n g p roduced some of the e a rliest genetic models of alcohol-re l a t e d b e h a v i o r s .
Individual differences among inbre d mice are theoretically due exc l u s i vely to e n v i ronmental factors, and when such factors are held constant, differe n c e s among inbred strains demonstrate genetic influence. The difficulty of holding environmental factors constant, howe ve r, was recently demonstrated in a study conducted by seve r a l d i f f e rent laboratories, in which a conc e rted effort was made to standard i ze the environment of the animals. Although the laboratories used animals obtained from the same sources and used the same testing pro c e d u res, they obtained differing results. The re s e a rchers concluded that only a stro n g genetic influence over a trait would negate the enviro nmental influences ( Crabbe et al. 1999a) . That is, if the mice are genetically identical, the diff e rences are due to unidentified environmental va r i a b l e s . Ne ve rtheless, inbred strains, part i c ularly strains of mice, have been successfully used to re veal genetic influences on responses to alcohol and to many other d rugs. The advantages of using inbre d strains include the ability to compare data collected in different laboratories (in spite of the environmental factors), the stability of strain responses over time, and the ability to compare acute and c h ronic responses to alcohol or other d rugs in different groups of mice. Di sa d vantages include the fact that it is difficult to generalize results from any one inbred strain to the mouse population as a whole (much less the human population) and that it is necessary to use a large number of inbred strains in o rder to generate valid genetic corre l ations. Howe ve r, many groups have made substantial pro g ress in investigating the genetics of alcohol-related behaviors using inbred strains.
Recombinant inbred (RI) strains are d e r i ved from a pair of standard inbre d strains. Within an inbred strain, each individual has two copies of the same form (i.e., allele) of each gene. Any two i n b red strains differ at some perc e n t a g e of the chromosome, randomly distributed across the complete set of chrom o s o m e s . The two progenitor strains a re interbred to generate a genetically identical F 1 population; at all areas of the chromosome where the pro g e n i t o r s d i f f e red, offspring re c e i ve one allele fro m each parent. Next, the F 1 s are randomly b red to produce the genetically heterogeneous F 2 population. In this population, parts of the parental chro m osomes have recombined. In b red strains a re generated from the F 2 p o p u l a t i o n by randomly chosen brother-sister mating for at least 20 generations. This i n b reeding produces animals that are genetically identical for one or the other p ro g e n i t o r's alleles at all locations on the chromosome. This process yields a unique pattern of recombinations of the parental chromosomes in each RI strain (Cr a b b e and Be l k n a p 1 9 9 2 ) .
The original use of this animal model was to provide a powe rful tool for establishing genetic associations and identifying major gene effects. Fo r example, if upon testing a group of RI strains for a response to alcohol, a re s e a rcher finds that the response of each RI strain resembles one of the re s p o n s e s of a pro g e n i t o r, this suggests that a single gene is exe rting an important influence. Once this major gene effect has been identified, linkage can be determined by matching the allele pattern for the locus (i.e., location on a chromosome) of the identified gene acro s s the RI strains (the "strain distribution p a t t e r n") with the strain distribution pattern for previously mapped genetic m a rkers. If the strain distribution patterns for the new locus and a mapped m a rker are the same, the new locus must be closely linked to the mark e r.
In addition to major gene effects, h owe ve r, RI strains can also be used to identify and locate genes that have smaller influences on the measure d trait. This is an important adva n c e , because most phenotypic characteristics (i.e., traits influenced by genetic factors), such as behavioral responses to alcohol, are not "all-or-none," but va ry over a continuous range of values. These q u a n t i t a t i ve traits are generally influenced by multiple genes (i.e., polygenic), and each gene may have only a small influence on the phenotype. In order to ident i f y the gene loci associated with quant i t a t i ve traits (i.e., quantitative trait loci [ QTL]), the allelic variation is corre l a t e d with the phenotypic variability in the RI strains. In other words, the behavioral or biochemical alcohol-related re s p o n s e s f rom the strains can be correlated with genetic markers, each scored as 0 or 1 to re p resent alleles from the two pro g e n itor strains. Significant correlations indicate associations between the mark e r ( s ) and the quantitative re s p o n s e s (i.e., a QTL). Fu rt h e r m o re, overlapping QT L s for two traits indicate genetic corre l ations between the traits. This appro a c h depends on the localization of a large number of markers on the genome and p rovides the starting point for identifying particular (candidate) genes within an identified QTL region. In animals, a re l a t i vely large number of QTLs for a l c o h o l -related behaviors have been identified (Crabbe et al. 1999b). The leap from QTL identification to gene identification will depend on the methodology for refining the position of the QTL (Rikke and Johnson 1998), which is currently being deve l o p e d .
The caveats associated with QT L analysis are essentially statistical, (e.g., the occurrence of false positives and false negatives). The level of powe r chosen to detect the statistical significance of the observed correlations allow s re s e a rchers to strike a balance betwe e n type I (i.e., false positive) and type II (i.e., false negative) errors. In general, QTLs detected using RI strains are c o n s i d e red "p rov i s i o n a l" and need to be confirmed. Confirmation may be accomplished, for example, by analysis of an F2 population, which can prov i d e a larger number of animals, and theref o re greater statistical strength, or by the use of congenic animals (i.e., inbre d animals that carry a small segment of a c h romosome from another strain).

Inbred strains have been successfully used
to reveal genetic influences on responses to alcohol and to many other drugs.
As markers on the human genome are mapped, QTL analysis can also be carried out in humans. Re c e n t l y, re s e a rchers have re p o rted several "susceptibility loci for alcohol dependence" in humans (Reich et al. 1998). Their identification is subject to the caveats noted above. The complex phenotype of alcoholism in humans and the polygenic influences on this disord e r suggest that genetic animal models will continue to provide crucial information for application to the human analysis. A c u r rent challenge is to create a categorization of behavioral-physiological corre l a t e s of alcoholism in humans for which animal models can provide both face and pre d i ct i ve va l i d i t y. Such categorization will provide for more re l e vant comparisons of genetic data obtained from humans and other animals.
Other strategies for evaluating a gene' s contribution to alcohol-related behaviors include the production of genetically e n g i n e e red transgenic and knock-out animals. These strategies, unlike QT L analysis, are not aimed at finding new genes, but at evaluating the import a n c e of candidate genes (genes believed to contribute to the development of a p a rticular disease). Candidate genes are generally identified through analyses of the neurochemical or biochemical determinants of alcohol-related re s p o n s e s . To c reate a transgenic mouse, a foreign gene is integrated into the mouse's own genetic material. Transgenic animals ove re x p re s s the foreign gene, and the influence of the gene on their responses to alcohol can be determined. This is a powe rful techn i q u e for assessing the influence of a candidate gene, although there are a number of c a veats associated with it. For example, u n t i l recently there has been no contro l over where in the chromosome the foreign gene, or transgene, is integrated. One way to circ u m vent this problem is to create a number of transgenic lines. Recently deve l o p e d techniques allow re s e a rchers to target the integration of the transgene. Er rors of i n t e r p retation can also occur if tissuespecific promoters are not used and the transgene is expressed in all tissues. As m o re tissue-specific promoters a re being identified, the alteration of gene e x p re s s i o n in specific cells and tissues will be facilitated. With respect to alcohol-re l a t e d behaviors and alcoholism, the influence of any single gene manipulation must be of a great enough magnitude that it can be reliably assessed. The issue of genetic b a c k g round is also import a n t , both for transgenic animals and the knock-out mice described below, because the magnitude of effects may va ry depending on the animal's own genetic makeup. The c reation of transgenic animals using several different backgrounds may prov i d e some control for this pro b l e m .
Knock-out mice are mice in which a gene has been inactivated or altere d . Studies with knock-out mice are subject to many of the problems mentioned for the transgenic mice, some of which are c u r rently being ove rcome as described. In addition, because some genes are essential for development, animals in which these critical genes are deleted do not surv i ve . In other cases, the gene mutation may p rompt compensatory adaptations during development that may ove rcome the effect of the alteration. These compens a t o ry changes, rather than the gene of i n t e rest, can there f o re influence the alcoh o l -related behaviors. Techniques to alleviate these problems include methods for conditional excision of the genes (i.e., conditional knock-outs), which could be p rogrammed to occur in the adult animal. Models are also being developed in which more subtle mutations, which c o n t rol the level of gene expression, can be introduced. Se veral knock-out mice h a ve been used in alcohol studies to date, including protein kinase C (PKC) and dopamine and serotonin (D 2 a n d 5 -H T 1 b ) receptor knock-outs, in which sensitivity to alcohol and alcohol drinki n g behavior have been assessed. Although these studies have re vealed some interesting differences between k n o c kout mice and wild-type (normal) mice, the differences have not always been replicable, perhaps reflecting the importance of compensatory adaptations during development as well as gene targeting and genetic backgro u n d .

Dose, Chronology, and Route of Administration Issues
In all animal models of alcohol-re l a t e d behaviors, investigators need to consider the alcohol doses used, the duration and spacing of alcohol administration, and the route of alcohol administration, in light of the influence of these parameters on the data obtained and its re l e vance to the human situation. For example, because the metabolic rate of alcohol varies among species, it is nece s s a ry to assure that the doses of alcohol used will generate blood and brain alcohol levels that can pro d u c e pharmacological effects. This has been a particular issue in studies using oral alcohol self-administration, where the amount of alcohol taken in may not e xceed the metabolic capacity of the animal and no significant accumulation of alcohol in brain or other tissues may o c c u r. In addition, different species have d i f f e rent sensitivities to the effects of alcohol. For example, mice appear to be less sensitive than rats to alcohol effects, such that similar doses (and similar brain and blood alcohol leve l s ) may produce different behavioral responses in the two species. This can be an important consideration when applying the results from animal model studies to humans. The use of end-points, such as the development of alcohol tolerance or physical dependence in animals, can provide a rationale for choosing a p a rticular dose or duration of alcohol e x p o s u re, even if the mode or duration of alcohol administration does not resemble that seen for humans.
The route of alcohol administration must also be considered. Feeding animals alcohol in a liquid diet as the sole s o u rce of nutrition differs from the normal pattern of alcohol intake in humans, even though alcohol is taken o r a l l y. Howe ve r, this method can be used reliably to generate tolerance, dependence, and the alcohol withdrawal effect. Si m il a r l y, models using continuous gast r i c infusion of ethanol are ve ry differe n t f rom the method of alcohol intake by humans. Howe ve r, by controlling for nutritional status and allowing high l e vels of alcohol intake, such models can be useful for understanding how alcohol ingestion can lead to organ damage. Operant techniques for oral self-administration of alcohol may or may not resemble the manner of alcohol intake by humans, and alcohol administration by injection or implanted tube is certainly different from the means of intake by humans. Even vo l u n t a ry alcohol intake in a two-bottle choice situation is quite different from human patterns of alcohol intake. Howe ve r, these models may provide insight into the neurophysiological basis for exc e ss i ve alcohol intake.

Conclusion
Thus, as already stated by Mc C l e a r n (1988), the experimental paradigms generated by re s e a rchers working with humans or animals are significantly constrained by the characteristics of the subjects, including the competence of a particular species to perform cert a i n tasks. Equipment and conceptual constraints may limit generalizability. The refinement of animal models needs to continue until the hypotheses arising f rom animal models can be ethically and practically tested on humans. s